Mechanisms of Chromosome Scale Signal Propagation

项目摘要 该项目的总体目标是确定细胞如何传达染色体断裂的形成， 在大的染色体距离上进行修复。DNA双链断裂（DSB）是一种危险的损伤， 基因组完整性，因为它们可能导致染色体重排和染色体不稳定性， 这两者都与癌症进展以及出生缺陷密切相关。值得注意的是，减数分裂 细胞能够有效地协调数百个并发DSB的形成和修复， 在减数分裂重组过程中，基因组的分裂是一个对配子形成和生育力至关重要的过程。 减数分裂DSB形成和修复的一个关键特征是其在染色体水平上的协调。在 在上一个资助期，我们提供了证据表明联会复合体，一种保守的蛋白质晶格， 在减数分裂后期的同源染色体之间形成，传达修复决定 沿着减数分裂染色体。啤酒。我们发现，这种沟通导致DSB减少， 形成以及简化的修复，我们确定了参与这一过程的几个因素。我们现在 发现在所有出现抗性的染色体末端附近存在特权基因组区域 由联会复合体调节。这些末端邻近区域（EARs）覆盖了大的基因组DNA， 距离（~ 100 kb，这是最短染色体长度的近一半），并继续形成和修复 在DSB形成后很久，DSB在基因组的其余部分停止。减数分裂的相似区域 在鸟类、黑猩猩和人类中也观察到了基因重组。 该项目的目标是确定产生这些区域的染色体信号，并测试是否 EAR有助于短染色体的遗传。我们的初步分析表明， 在基因组的其余部分中，EAR的建立和DSB的抑制都是由基因包封引起的。 染色体信号传导的动力学及其与核膜的相互作用将通过 全基因组结合研究和超分辨率显微镜，利用条件核 我们最近引入到减数分裂细胞中，允许阶段特异性敲低 多效性核因子此外，将使用遗传上位性分析来分析信号整合， 细胞学和DSB形成的物理分析。由于EAR覆盖了比例上大得多的空头比例， 该提案还将使用四分体测序来测试这些区域是否驱动广泛观察到的 短染色体上重组率增加。荧光标记分离将用于 确定EAR是否差异性地改善短染色体的减数分裂分离保真度。所有这些 分析将提供染色体信号传播机制的关键见解，并开辟新的 了解出生缺陷起源的途径，如唐氏综合症（21三体）和爱德华兹 综合征（18三体），其由短染色体的减数分裂错误分离引起。

Project summary The overall goal of this project is to determine how cells communicate chromosome break formation and repair across large chromosomal distances. DNA double-strand breaks (DSBs) are dangerous insults to genome integrity because of their potential to cause chromosome rearrangements and chromosome instability, both of which are strongly associated with cancer progression as well as birth defects. Remarkably, meiotic cells are able to efficiently orchestrate the formation and repair of hundreds of concurrent DSBs across their genome during meiotic recombination, a process that is essential for proper gamete formation and fertility. A key feature of meiotic DSB formation and repair is its coordination at the chromosomal level. In the previous funding period we provided evidence that the synaptonemal complex, a conserved protein lattice that forms between aligned homologous chromosomes in late meiotic prophase, communicates repair decisions along meiotic chromosomes in S. cerevisiae. We showed that this communication resulted in reduced DSB formation as well as simplified repair, and we identified several factors involved in this process. We now discovered the existence of privileged genomic regions near the ends of all chromosomes that appear resistant to regulation by the synaptonemal complex. These end-adjacent regions (EARs) cover large genomic distances (~100kb, which is nearly half the length of the shortest chromosome) and continue to form and repair DSBs well after DSB formation has stopped in the rest of the genome. Similar regions of elevated meiotic recombination are also observed in birds, chimps, and humans. The goal of this project is to define the chromosomal signal that generates these regions and to test if EARs help inheritance of short chromosomes. Our preliminary analyses suggest several roles of the nuclear envelope, both in the establishment of the EARs and in the suppression of DSBs in the rest of the genome. The dynamics of chromosomal signaling and its interaction with the nuclear envelope will be analyzed by genome-wide binding studies and super-resolution microscopy, taking advantage of a conditional nuclear depletion approach that we recently introduced into meiotic cells that allows stage-specific knock-downs of pleiotropic nuclear factors. In addition, signal integration will be analyzed using genetic epistasis analyses, cytology, and physical analysis of DSB formation. As EARs cover a proportionally much larger fraction of short chromosomes, the proposal will also use tetrad sequencing to test if these regions drive the widely observed increase in recombination rates on short chromosomes. Fluorescent marker segregation will be used to determine if EARs differentially improve the meiotic segregation fidelity of short chromosomes. Together, these analyses will provide key insights into the mechanisms of chromosomal signal propagation, and open new avenues for understanding the origins of birth defects such as Down syndrome (trisomy 21) and Edwards syndrome (trisomy 18), which are caused by meiotic missegregation of short chromosomes.